A Study On Methylation Difference Between Human Large Cell Lung Cancer Cell Lines With Different Metastatic Potential

Background and Object Invasion and metastasis is onemalignant phenotype of lung cancer and cause the death of lung cancer patients. Present evidence has proved that invasion and metastasis in lung cancer is a complicated course with multiple steps and stages and regulated both by metastasis suppressor genes and metastasis promoting genes. Therefore, it is very important to study and elucidate the molecular mechanism of invasion and metastasis in lung cancer. Nm23-Hl was originally identified as a tumor metastasis suppressor gene. The abnormality of its function and structure was closely correlated with the invasion and metastasis of cancer cells. Our previous research has proved that the reduced expression and hetero-deletion of nm23-H1 gene is closely correlated with the high metastasis potential of lung cancer and the key upstream gene of "metastasis suppress cascades". Although little is known about the exact anti-metastasis molecular mechanisms of nm23-H1. Resently, the study of epigenic becomes hot in oncology fields. Epigenic refers to the study of heritable changes in gene expression that occur without a change in DNA sequence. The change of the phenotype without alteration of the genotype can be transmitted by cell proliferation. DNA methylation is an important part of epigenic, which plays an important role in normal cell function maintaining, genetic imprinting, embryonic development, and closely related to human tumorigenesis. The hypermethylation of tumor suppressing gene and the hypomethylation of oncogene is one important research in oncology field. Our research aims to compare the DNA methylation of two large cell lung cancer cell lines with different metastatic potential by CpG island chip hybridization to settle a foundation for further research of gene methylation effection on lung cancer metastasis.Materials and Methods (1) The genome DNA of L9981 andNL9980 cell lines was extracted by TIANamp Genomic DNA kit and sonicated into fragments by sonicator;(2) The genome DNA fragments were immunoprecipitated by MBD2b ;(3)The immunoprecipitated DNA fragments were amplified by LM-PCR;(4) The DNA fragments were labled with cy3 and cy5 by direct oligonucleotide incorporation and hybridizated with a high through CpG chip;(5) SBC human CHIP was scanned by GENEPIX4000B scanner;(6)The difference of gene methylation between L9981 and NL9980 was analyzed by nimblescan;(7) Hypermethylated and hypomethylated genes of L9981 cell lines were analyzed online in NIH-DAVID and MILANO webside.Results (l)Compared with NL9980, 97 hypomethylated genes in L9981 were found, including 25 known genes and 52 unknown gene; (2) Compared with NL9980, 353 hypermethylated genes in L9981 were found, including 67 known genes and 353 unknown genes; (3) 1 gene (PXMP2) among the 25 known hypomethylated genes in L9981 were reported related to metastasis, 2 genes (PXMP2, HYAL1) related to invasion, 4 related to apoptosis, 5 related to cell cycle; (4) 4 gene among the 67 known hypermethylated genes in L9981 were reported related to metastasis, 3 genes related to invasion, 12 related to apoptosis,Conclusion Tthe difference of the DNA methylation was analyzedbetween two large cell lung cancer cell lines with different metastatic potential, and 617 genes with different methylation were found, which mainly involved in metastsis, invasion, apoptosis and cell cycle regulation. A foundation was settled for further research on effection of gene methylation on lung cancer metastasis.